Biotinylation fluorescent derivatives

Other avidin-enzyme conjugates that convert solid substrates to colored precipitates can be used. Likewise, fluorescent derivatives of avidin can be used, and the sample can be viewed with a fluorescent microscope two different fluorophores can be employed in double-labeling experiments (ieethis vol., Chapters 11 and 42). Preformed complexes containing avidin and biotinylated enzyme can also be used in place of the covalently coupled conjugate. 

A phosphoramidite monomer derived from (71) has been introduced into ODNs for post-synthesis modification. The ketone was treated with a number of aminooxy derivatives, and it was shown that the modification had little effect on duplex stability. This may be used to decorate DNA for a number of DNA applications. A series of modified dUTP analogues (72) which can incorporate a variety of amine derivatives, including biotinylated and fluorescent derivatives, has been prepared for study in PCR reactions. It was found that KOD Dash DNA polymerase was able to incorporate the modified triphosphates during PCR whilst other conventional DNA polymerases would not. 

Kuramochi, K., Yukizawa, S., Ikeda, S., et al. (2008). Syntheses and applications of fluorescent and biotinylated epolactaene derivatives Epolactaene and its derivative induce disulfide formation. Bioorg. Med. Chem., 16, 5039-5049. 

Also recently, Liao and collaborators [89] proposed a homogeneous noncompetitive assay of a protein in biological samples based on FRET by using its tryptophan residues as intrinsic donors and its specific fluorescent ligand as the FRET acceptor, which was defined as an analytical FRET probe. To evaluate this method, a naphthylamine derivative, namely /V-biotinyl-/V -(l -naphthylj-ethylene-diamine (BNEDA) 33 was used as an analytical FRET probe for the homogeneous noncompetitive assay of streptavidin. 

The spectral properties of four major phycobiliproteins used as fluorescent labels can be found in Tables 9.1 and 9.2. The bilin content of these proteins ranges from a low of four prosthetic groups in C-phycocyanin to the 34 groups of B- and R-phycoerythrin. Phycoerythrin derivatives, therefore, can be used to create the most intensely fluorescent probes possible using these proteins. The fluorescent yield of the most luminescent phycobiliprotein molecule is equivalent to about 30 fluoresceins or 100 rhodamine molecules. Streptavidin-phycoerythrin conjugates, for example, have been used to detect as little as 100 biotinylated antibodies bound to receptor proteins per cell (Zola et al., 1990). 

Other fluorescent probes also may be used to label (strept)avidin molecules for detection of biotinylated targeting molecules. Chapter 9 reviews many additional fluorescent labels, such as quantum dots, lanthanide chelates, and cyanine dye derivatives, all of which may be used in similar protocols to create detection conjugates for (strept)avidin-biotin-based assays. 

The use of this reaction in the biological context was first demonstrated for the chemospecific labeling of Jurkat cell surfaces [63]. Metabolic engineering with N-acetylmannosamine derivative 40c was used to incorporate azides into sialic acid groups on cell surfaces. The cells were then incubated with biotinylated phosphine 49, and the extent of the reaction was quantified by flow cytometry after treatment with fluorescent avidin. Importantly, neither the azide nor the phosphine displayed any reactivity with the cell-surface groups in the absence of its reactive partner. In addition, the cells showed unchanged growth rates after modification. 

First, examples of fluorescence [235] and chemiluminescence-based biosensors [236], derived fix)m PTs, were reported by Tripathy and coworkers. Later, the synthesis of 119 containing pendant biotin units was described. A water-soluble copolymer 120 with sulfonate and biotin in the side chains [237] exhibits a deep violet color (Amax = 550 run) which turns yellow (A ax = 400 nm) on binding with avidin. An extension of this work based on the homopolymer 121 involved the preparation of monolayers of a biotinylated PT on an aminosilane-treated ITO surface by successive deposition of 121 and biocytin hydrazide in electrostatic interactions [238,239]. This ultrathin film modified electrode was shown to detect femtomoles of avidin in aqueous solution. Electrochemical and optical evidences for avidin binding were reported for a copolymer based on poly(terthiophene) 122 [240] and for the homopolymer 123 [198]. 

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